Polypeptides of the maize amyloplast stroma. Stromal localization of starch-biosynthetic enzymes and identification of an 81-kilodalton amyloplast stromal heat-shock cognate

Abstract

In the developing endosperm of monocotyledonous plants, starch granules are synthesized and deposited within the amyloplast. A soluble stromal fraction was isolated from amyloplasts of immature maize (Zea mays L.) endosperm and analyzed for enzyme activities and polypeptide content. Specific activities of starch synthase and starch-branching enzyme (SBE), but not the cytosolic marker alcohol dehydrogenase, were strongly enhanced in soluble amyloplast stromal fractions relative to soluble extracts obtained from homogenized kernels or endosperms. Immunoblot analysis demonstrated that starch synthase I, SBEIIb, and sugary1, the putative starch-debranching enzyme, were each highly enriched in the amyloplast stroma, providing direct evidence for the localization of starch-biosynthetic enzymes within this compartment. Analysis of maize mutants shows the deficiency of the 85-kD SBEIIb polypeptide in the stroma of amylose extender cultivars and that the dull mutant lacks a >220-kD stromal polypeptide. The stromal fraction is distinguished by differential enrichment of a characteristic group of previously undocumented polypeptides. N-terminal sequence analysis revealed that an abundant 81-kD stromal polypeptide is a member of the Hsp70 family of stress-related proteins. Moreover, the 81-kD stromal polypeptide is strongly recognized by antibodies specific for an Hsp70 of the chloroplast stroma. These findings are discussed in light of implications for the correct folding and assembly of soluble, partially soluble, and granule-bound starch-biosynthetic enzymes during import into the amyloplast.

Figure 1

Effect of Triton X-100 on SS recovery and activity. A, SS levels of amyloplast stromal fractions. Amyloplasts were released from endosperm, as described in Methods and recovered by low-speed centrifugation. Stromal fractions were generated by suspension of amyloplasts in buffer A containing Triton X-100 at the levels indicated. Suspensions were mixed and centrifuged. Stromal fractions were recovered and assayed for SS activity. B, Effect of Triton X-100 on AGP (▴), SBE (•), and SS (□) activities of a soluble endosperm extract. Endosperm extracts were prepared as described in Methods and aliquots were assayed at the indicated levels of Triton X-100.

Figure 2

Enrichment of SS and SBE in amyloplast stromal extracts. Specific activities of SS (A; nanomoles per minute per milligram), SBE (B; micromoles per minute per milligram), ADH, the cytosolic marker enzyme (C; micromoles per minute per milligram), and AGP assayed with 10 mm 3-phosphoglyceric acid (D; micromoles per minute per milligram). Solid and striped bars represent tissue harvested at 13 and 15 DAP, respectively. Values are representative of two independent isolations.

Figure 3

Immunoblots of endosperm and amyloplast stromal extracts probed with ADH, SSI, SBEIIb, and SU1 antibodies. SDS gels were run with amyloplast stromal fractions (Am), soluble extracts from whole endosperm (En), or SDS extracts corresponding to 2.5 mg of isolated starch granules (Gr). Immunoblots were probed with antibodies recognizing ADH (A), SSI (B), SBEIIb (C), and SU1 (D). The blots shown in A and D were visualized by electrochemiluminescence. The blots shown in B and C were visualized colorimetrically. Each lane contained 5 μg of protein.

Figure 4

Immunoblots of endosperm and amyloplast stromal extracts probed with SH2 and BT2 antibodies. SDS gels were run with amyloplast stromal fractions (Am) or soluble extracts from whole endosperm (En). Immunoblots were probed with antibodies recognizing SH2, the AGP large subunit, or BT2, the AGP small subunit, as indicated. Each lane contained 5 μg of protein.

Figure 5

Protein composition of soluble extracts from whole endosperm and isolated amyloplasts. A, Coomassie blue-stained gel with 15 μg of protein per lane. B, Silver-stained gel with 10 μg of protein per lane. Shown are the soluble extracts from whole ground endosperm (En) and the amyloplast stromal fraction (Am). Marks indicate stromal proteins in descending order of apparent molecular mass: >220, 215, 180, 134, 112, 107, 85, 81, and 76 kD.

Figure 6

N-terminal sequences of SBEIIb (85 kD; A) and the stromal Hsc70 (81 kD; B). Polypeptides were excised from SDS gels and electropurified as previously described (Mu-Forster et al., 1996). The dash at position 16 indicates an uncertain residue tentatively identified as an Arg residue. Published sequences were as follows: SBEIIb beginning at residue 53 of the predicted sequence (Fisher et al., 1993); chloroplast Hsp70 from Pisum sativum (Marshall and Keegstra, 1992); chloroplast Hsp70 from Curcurbita sp. (Tsugeki and Nishimura, 1993); chromoplast Hsp70 from Narcissus pseudonarcissus (Bonk et al., 1996); mitochondrial SSC1, an Hsp70 analog from Saccharomyces cerevisiae (Craig et al., 1989); dnaK, an Hsp70 analog from E. coli (Bardwell and Craig, 1984); and cytosolic Hsp70 from Z. mays endosperm (Rochester et al., 1986).

Figure 7

Immunoblots of endosperm and amyloplast stromal extracts probed with chloroplast Hsp70 antibody. Lane 1, Hsp70 preparation containing two closely migrating Hsp70 proteins purified from E. gracilis (Amir-Shapira et al., 1990; 0.1 μg); lane 2, soluble extract from whole endosperm (En; 30 μg); lane 3, amyloplast stromal fraction (Am; 30 μg); and lane 4, purified maize stromal 81-kD polypeptide (0.1 μg).

Figure 8

Polypeptide profiles of amyloplast stroma of amylose extender (ae) and dull (du) mutants. Stroma are from an Exs86 parental line (lane 1), an isogenic dull mutant (lane 2), and an amylose extender mutant (lane 3). Each lane contained 30 μg of protein.

Figure 9

Effect of Triton X-100 on the extraction of soluble polypeptides from suspensions of whole endosperms and isolated amyloplasts. Lanes 1 and 2, Soluble fraction from whole endosperm; lanes 3 and 4, soluble fraction from isolated amyloplasts. Soluble extracts were prepared in buffer A in the presence (+) or absence (−) of 0.3% Triton X-100. Each lane contained 25 μg of protein.

Figure 10

Working model. A proposed mechanism for the import and translocation of polypeptides into the amyloplast stromal compartment illustrating a possible mode of action of the 81-kD Hsc70. This model proposes that chaperones of the Hsp70 class facilitate the import and folding of enzymes that are fully (I) or partially (II) soluble within the stroma. A proposed function of the chaperones is to stabilize the partially soluble mature proteins, delaying their insolubilization within the starch matrix. A separate route for the import and deposition of fully insoluble (e.g. GBSSI) amyloplast proteins (III) is shown.